Cystic fibrosis (CF) is a fatal disease of abnormal epithelial ion- transport, characterized principally by reduced Cl- and increased amiloride-sensitive Na+ conductances in pulmonary epithelia. Since the gene coding for the cystic fibrosis conductance regulator (CFTR) was identified, intense efforts have been directed toward understanding the malfunction of Cl- secretion. However, the abnormalities associated with Na+ transport are poorly understood. Recent identification of alpha, beta and gamma subunits that are presumed to constitute the functional epithelial Na+ channel (ENaC) makes possible the mechanistic studies of the abnormally raised and cAMP regulated Na+ transport associated with CF. In heterologous systems, ENaC activity is high and stimulated by cAMP, whereas, in the presence of CFTR, ENaC is down- regulated and cAMP effects reversed mimicking the in vivo observations of CF and normal airway epithelia, respectively. These observations form the basis for the long term goal of this grant application. Two heterologous expression systems, MDCK and Sf9 insect cells expressing ENaC and CFTR, will be utilized to undertake a detailed biochemical investigation of the structure and regulation of the ENaC. Utilizing these experimental systems, a research plan has been organized around 3 specific aims. 1. We shall isolate the functional ENaC and determine its composition and stoichiometry of the subunits. 2. We shall investigate the mechanism by which cAMP stimulates the ENaC. We shall identify the amino acid residues that are phosphorylated in the ENaC, and correlate the phosphorylation events to the function by site- directed mutagenesis. 3. We shall determine the mechanism by which CFTR regulates ENaC function. We shall investigate the hypothesis that CFTR regulates the ENaC by the mechanisms involving phosphorylation and dephosphorylation of amino acids in the ENaC subunits. We shall also test the hypothesis that physical interactions of CFTR with ENaC are responsible for down-regulation of ENaC function by immunological methods. It is anticipated that this research will aid further in understanding the ENaC-mediated Na+ absorption process across the reabsorbing epithelia and ameliorating the diseases associated with ENaC dysfunction.